ABSTRACT
BACKGROUND: Angiogenesis is involved in tumor growth, macular degeneration, retinopathy and other diseases. Vascular endothelial growth factor (VEGF) stimulates angiogenesis by binding to specific receptors (VEGFRs) on the surface of vascular endothelial cells. VEGFRs are receptor tyrosine kinases that, like the platelet-derived growth factor receptors (PDGFRs), contain a large insert within the kinase domain. RESULTS: We report here the generation, kinetic characterization, and 2.4 A crystal structure of the catalytic kinase domain of VEGF receptor 2 (VEGFR2). This protein construct, which lacks 50 central residues of the 68-residue kinase insert domain (KID), has comparable kinase activity to constructs containing the entire KID. The crystal structure, determined in an unliganded phosphorylated state, reveals an overall fold and catalytic residue positions similar to those observed in other tyrosine-kinase structures. The kinase activation loop, autophosphorylated on Y1059 prior to crystallization, is mostly disordered; however, a portion of it occupies a position inhibitory to substrate binding. The ends of the KID form a beta-like structure, not observed in other known tyrosine kinase structures, that packs near to the kinase C terminus. CONCLUSIONS: The majority of the VEGFR2 KID residues are not necessary for kinase activity. The unique structure observed for the ends of the KID may also occur in other PDGFR family members and may serve to properly orient the KID for signal transduction. This VEGFR2 kinase structure provides a target for design of selective anti-angiogenic therapeutic agents.
Subject(s)
Neovascularization, Physiologic , Protein Conformation , Receptor Protein-Tyrosine Kinases/chemistry , Receptors, Growth Factor/chemistry , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Growth Substances/chemistry , Humans , Kinetics , Models, Molecular , Molecular Sequence Data , Multigene Family , Mutagenesis, Site-Directed , Peptide Fragments/chemistry , Protein Folding , Protein Structure, Secondary , Protein-Tyrosine Kinases/chemistry , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/physiology , Receptors, Growth Factor/genetics , Receptors, Growth Factor/physiology , Receptors, Vascular Endothelial Growth Factor , Recombinant Fusion Proteins/chemistry , Sequence Alignment , Sequence Homology, Amino Acid , Structure-Activity Relationship , Substrate SpecificityABSTRACT
The structure-based design and subsequent chemical synthesis of novel, urea-containing FKBP12 inhibitors are described. These compounds are shown to disrupt the cis-trans peptidylprolyl isomerase activity of FKBP12 with inhibition constants (Ki,app) approaching 0.10 microM. Analyses of several X-ray crystal structures of FKBP12-urea complexes demonstrate that the urea-containing inhibitors associate with FKBP12 in a manner that is similar to, but significantly different from, that observed for the natural product FK506.
Subject(s)
Carrier Proteins/antagonists & inhibitors , DNA-Binding Proteins/antagonists & inhibitors , Drug Design , Heat-Shock Proteins/antagonists & inhibitors , Urea/analysis , Amino Acid Isomerases/antagonists & inhibitors , Amino Acid Sequence , Carrier Proteins/chemistry , Crystallography, X-Ray , DNA-Binding Proteins/chemistry , Heat-Shock Proteins/chemistry , Humans , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Peptidylprolyl Isomerase , Structure-Activity Relationship , Tacrolimus/chemistry , Tacrolimus Binding ProteinsABSTRACT
Calcineurin (CaN) is a calcium- and calmodulin-dependent protein serine/threonine phosphate which is critical for several important cellular processes, including T-cell activation. CaN is the target of the immunosuppressive drugs cyclosporin A and FK506, which inhibit CaN after forming complexes with cytoplasmic binding proteins (cyclophilin and FKBP12, respectively). We report here the crystal structures of full-length human CaN at 2.1 A resolution and of the complex of human CaN with FKBP12-FK506 at 3.5 A resolution. In the native CaN structure, an auto-inhibitory element binds at the Zn/Fe-containing active site. The metal-site geometry and active-site water structure suggest a catalytic mechanism involving nucleophilic attack on the substrate phosphate by a metal-activated water molecule. In the FKBP12-FK506-CaN complex, the auto-inhibitory element is displaced from the active site. The site of binding of FKBP12-FK506 appears to be shared by other non-competitive inhibitors of calcineurin, including a natural anchoring protein.
Subject(s)
Adaptor Proteins, Signal Transducing , Calmodulin-Binding Proteins/chemistry , Carrier Proteins/metabolism , DNA-Binding Proteins/metabolism , Heat-Shock Proteins/metabolism , Phosphoprotein Phosphatases/chemistry , Tacrolimus/metabolism , A Kinase Anchor Proteins , Amino Acid Sequence , Binding Sites , Calcineurin , Calcium/metabolism , Calmodulin-Binding Proteins/antagonists & inhibitors , Calmodulin-Binding Proteins/metabolism , Calmodulin-Binding Proteins/ultrastructure , Carrier Proteins/chemistry , Crystallization , Crystallography, X-Ray , DNA-Binding Proteins/chemistry , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Heat-Shock Proteins/chemistry , Humans , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphoprotein Phosphatases/metabolism , Phosphoprotein Phosphatases/ultrastructure , Protein Conformation , Protein Structure, Secondary , Proteins/metabolism , Proteins/pharmacology , Recombinant Proteins/chemistry , Tacrolimus/chemistry , Tacrolimus Binding Proteins , Water/metabolismABSTRACT
Density-dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of an extracellular signal molecule (autoinducer) in the culture medium. A recombinant clone that restored function to one class of spontaneous dim mutants was found to encode functions necessary for the synthesis of, and response to, a signal molecule. Sequence analysis of the region encoding these functions revealed three open reading frames, two (luxL and luxM) that are required for production of an autoinducer substance and a third (luxN) that is required for response to this signal substance. The LuxL and LuxM proteins are not similar in amino acid sequence to other proteins in the database, but the LuxN protein contains regions of sequence resembling both the histidine protein kinase and the response regulator domains of the family of two-component, signal transduction proteins. The phenotypes of mutants with luxL, luxM and luxN defects indicated that an additional signal-response system controlling density-dependent expression of luminescence remains to be identified.
Subject(s)
Aldehyde Oxidoreductases/genetics , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Genes, Bacterial , Luciferases/genetics , Luminescent Measurements , Operon , Protein Kinases , Repressor Proteins , Signal Transduction , Trans-Activators , Transcription Factors , Vibrio/physiology , Amino Acid Sequence , Bacterial Proteins/metabolism , Base Sequence , Cloning, Molecular , Consensus Sequence , Homoserine/analogs & derivatives , Homoserine/biosynthesis , Molecular Sequence Data , Mutagenesis, Insertional , Open Reading Frames , Phenotype , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Vibrio/geneticsABSTRACT
Mutagenesis with transposon mini-Mulac was used previously to identify a regulatory locus necessary for expression of bioluminescence genes, lux, in Vibrio harveyi (M. Martin, R. Showalter, and M. Silverman, J. Bacteriol. 171:2406-2414, 1989). Mutants with transposon insertions in this regulatory locus were used to construct a hybridization probe which was used in this study to detect recombinants in a cosmid library containing the homologous DNA. Recombinant cosmids with this DNA stimulated expression of the genes encoding enzymes for luminescence, i.e., the luxCDABE operon, which were positioned in trans on a compatible replicon in Escherichia coli. Transposon mutagenesis and analysis of the DNA sequence of the cloned DNA indicated that regulatory function resided in a single gene of about 0.6-kilobases named luxR. Expression of bioluminescence in V. harveyi and in the fish light-organ symbiont Vibrio fischeri is controlled by density-sensing mechanisms involving the accumulation of small signal molecules called autoinducers, but similarity of the two luminescence systems at the molecular level was not apparent in this study. The amino acid sequence of the LuxR product of V. harveyi, which indicates a structural relationship to some DNA-binding proteins, is not similar to the sequence of the protein that regulates expression of luminescence in V. fischeri. In addition, reconstitution of autoinducer-controlled luminescence in recombinant E. coli, already achieved with lux genes cloned from V. fischeri, was not accomplished with the isolation of luxR from V. harveyi, suggesting a requirement for an additional regulatory component.
